23. The insulin preparation of claim 22, wherein the nicotinic compound
is nicotinamide.

24. The insulin preparation of claim 23 wherein the nicotinamide is
present at a concentration ranging from about 80 mM to about 260 mM.

25. The insulin preparation of claim 24, wherein the arginine is present
in a concentration ranging from about 10 mM to about 40 mM.

26. The insulin preparation of claim 25, wherein the preservative is
selected from the group consisting of phenol, cresol, and combinations
thereof, and wherein the preparation has a pH of about 7.4 or less.

27. The insulin preparation of claim 26, wherein less than about 4 zinc
ions are present per six insulin analog molecules.

28. The insulin preparation of claim 26, wherein the preparation has a pH
of about 7.1

[0002] The present invention relates to pharmaceutical preparations
comprising an insulin compound, a nicotinic compound and an amino acid.

BACKGROUND OF THE INVENTION

[0003] Diabetes mellitus is a metabolic disorder in which the ability to
utilize glucose is partly or completely lost. About 5% of all people
suffer from diabetes and the disorder approaches epidemic proportions.

[0004] Since the introduction of insulin in the 1920's, continuous
improvements have been made in the treatment of diabetes. To help avoid
high glycaemia levels, diabetic patients often practice multiple
injection therapy, whereby insulin is administered with each meal. As
diabetic patients have been treated with insulin for several decades,
there is a major need for safe and life-quality improving insulin
preparations. Among the commercially available insulin preparations,
rapid-acting, intermediate-acting and prolonged-acting preparations can
be mentioned.

[0005] In the treatment of diabetes mellitus, many varieties of
pharmaceutical preparations of insulin have been suggested and used, such
as regular insulin (such as Actrapid®), isophane insulin (designated
NPH), insulin zinc suspensions (such as Semilente®, Lente®, and
Ultralente®), and biphasic isophane insulin (such as NovoMix®).
Human insulin analogues and derivatives have also been developed,
designed for particular profiles of action, i.e. fast action or prolonged
action. Some of the commercially available insulin preparations
comprising such rapid acting insulin analogues include NovoRapid®
(preparation of B28Asp human insulin), Humalog® (preparation of
B28LysB29Pro human insulin) and Apidra® (preparation of B3LysB29Glu
human insulin).

[0007] Most often pharmaceutical preparations of insulins are administered
by subcutaneous injection. Important for the patient is the action
profile of the insulin, meaning the action of insulin on glucose
metabolism as a function of time from injection. In this profile, inter
alia, the time of the onset, the maximum value and the total duration of
action are important. In the case of bolus insulins, a variety of insulin
preparations with different action profiles are desired and requested by
the patients. One patient may, on the same day, use insulin preparations
with very different action profiles. The action profile desired for
example, depends on the time of the day and the amount and composition of
the meal eaten by the patient.

[0008] Equally important for the patient is the chemical stability of the
insulin preparations, for example, due to the abundant use of pen-like
injection devices such as devices which contain Penfill® cartridges,
in which an insulin preparation is stored until the entire cartridge is
empty which may be at least 1 to 2 weeks for devices containing 1.5-3.0
ml cartridges. During storage, covalent chemical changes in the insulin
structure occur. This may lead to formation of molecules which may be
less active and/or potentially immunogenic such as deamidation products
and higher molecular weight transformation products (dimers, polymers).
Furthermore, also important is the physical stability of the insulin
preparations, since long term storage may eventually lead to formation of
insoluble fibrils, which are biologically inactive and potentially
immunogenic.

[0010] In one embodiment, the present invention relates to an insulin
preparation comprising:

[0011] an insulin compound,

[0012] a nicotinic compound, and

[0013] arginine.

In another embodiment the insulin preparation may further comprise
glutamic acid.

[0014] In another embodiment, the present invention also contemplates a
method for the treatment of diabetes mellitus in a subject or for
reducing the blood glucose level in a subject comprising administering to
a subject or mammal an insulin preparation according to the invention.

DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the development in percentage of total insulin content
of degradation products during 2 weeks of storage at 37° C. of
preparations according to the present invention. The letter A refers to a
NovoRapid® reference and remaining letters correspond to insulin
aspart preparations as described in Table 1 of Example 1. Compared to the
NovoRapid® preparation (preparation A), addition of nicotinamide
(preparations B and D) leads to an increased formation of degradation
products, whereas the combined addition of nicotinamide, glutamic acid
and arginine (preparations C and E), has a mostly similar degradation
pattern, with lower formation of HMWP.

[0016]FIG. 2 shows the development in percentage of total insulin content
of degradation products during 2 weeks of storage at 37° C. of
preparations according to this invention. The letter A refers to a
NovoRapid® reference and remaining letters correspond to insulin
aspart preparations as described in Table 1 of Example 1. The combined
addition of nicotinamide, glutamic acid and arginine, preparations F, G,
H, and I, differing in buffer system, phosphate or tris buffer, and
concentration of insulin and Zn, 0.6 mM and 0.3 mM or 1.2 mM and 0.6 mM,
has a degradation pattern similar to the NovoRapid® preparation,
preparation A.

[0017]FIG. 3 shows the glucose concentration (mean+/-SEM, N=8) in plasma
after subcutaneous injection in pigs of a 1 nmol/kg dose at 0 minutes of
preparations according to this invention. The letter A refers to a
NovoRapid® reference and remaining letters correspond to insulin
aspart preparations as described in Table 1 of Example 1. Compared to the
NovoRapid® preparation (preparation A) the initial rate of plasma
glucose lowering is faster for the preparation with addition of
nicotinamide (preparation N) and even faster for a combination of
nicotinamide and arginine (preparation M).

[0018]FIG. 4 shows the glucose concentration in plasma (mean+/-SEM, N=7)
after subcutaneous injection in pigs of a 1 nmol/kg dose at 0 minutes of
preparations according to this invention. The letter A refers to a
NovoRapid® reference and remaining letters correspond to insulin
aspart preparations as described in Table 1 of Example 1. Compared to the
NovoRapid® preparation (preparation A), the initial rate of plasma
glucose lowering is faster for a preparation with a combination of
nicotinamide, arginine and glutamic acid (preparation L) and for a
preparation with a combination of nicotinamide and arginine (preparation
K).

[0019]FIG. 5 shows the insulin aspart concentration in plasma
(mean+/-SEM, N=7) after subcutaneous injection in pigs of a 1 nmol/kg
dose at 0 minutes of preparations according to this invention. The letter
A refers to a NovoRapid® reference and remaining letters correspond
to insulin aspart preparations as described in Table 1 of Example 1.
Compared to the NovoRapid® preparation, (preparation A), the initial
absorption rate of the insulin component of the preparations with
nicotinamide (preparation J), the combination of nicotinamide and
arginine (preparation K), and the combination of nicotinamide, arginine
and glutamic acid (preparation L) is markedly faster.

[0020]FIG. 6 shows the time course for fibril formation can be described
by a sigmoidal curve using Equation 1.

DESCRIPTION OF THE INVENTION

[0021] The absorption after subcutaneous injection of the insulin compound
in the insulin preparations of the present invention was surprisingly
found to be faster than that of the reference insulin preparations. This
property is useful for rapid-acting insulins, in particular in connection
with a multiple injection regimen where insulin is given before each
meal. With faster onset of action, the insulin can conveniently be taken
closer to the meal than with conventional rapid acting insulin solutions.
Furthermore, a faster disappearance of insulin probably diminishes the
risk of post-meal hypoglycaemia.

[0022] The insulin preparations of the present invention are rapid-acting
insulin preparations comprising an insulin compound such as insulin
aspart, a nicotinic compound, such as nicotinamide and the amino acid
arginine. Optionally, the insulin preparations of the present invention
may comprise further amino acids such as glutamic acid. These insulin
preparations have a rapid absorption profile that mimics normal
physiology more closely than existing therapies. Furthermore, the insulin
preparations of the present invention have chemical and physical
stability suitable for commercial pharmaceutical preparations.

[0023] The insulin preparations of the present invention provide an even
faster onset of action compared with existing insulin therapies. Such
ultra-fast insulin preparations have the advantage of restoring first
phase insulin release, injection convenience and shutting down hepatic
glucose production. The insulin preparations of the present invention
have a favourable absorption rate from subcutis into plasma with an
increase in initial absorption rate ranging from 1.5 to 5 times, when
compared to conventional preparations such as NovoRapid®, as
suggested by several PK/PD experiments in pigs. This faster absorption
rate may improve glycaemic control and convenience and may allow for a
shift from pre-meal to post-meal dosing. The present invention is based
in part, on the surprising discovery that although, the addition of
nicotinamide allows the increase in absorption rate, it also has a
negative effect on chemical stability by significantly increasing the
amount of HMWP. The insulin preparations of the present invention have an
improved chemical stability by addition of arginine, which is reflected
in e.g. a reduction in the formation of dimers and polymers and desamido
insulins after storage. The insulin preparations of the present invention
may furthermore also have improved physical stability, which may be
useful for use in pumps.

[0024] The present invention provides an insulin preparation comprising an
insulin compound according to the present invention which is present in a
concentration from about 0.1 mM to about 10.0 mM, and wherein said
preparation has a pH from 3 to 8.5. The preparation also comprises a
nicotinic compound and arginine. The preparation may further comprise
protease inhibitor(s), metal ions, a buffer system, preservative(s),
tonicity agent(s), chelating agent(s), stabilizers and surfactants.

[0026] In one embodiment, the insulin preparations according to the
present invention comprise an aqueous solution of B28Asp human insulin,
nicotinamide and arginine.

[0027] The content of B28Asp human insulin in the solutions of this
invention may be in the range of 15 to 500 international units (IU)/ml,
preferably in the range of 50 to 333 IU/ml, in preparations for
injection. However, for other purposes of parenteral administration, the
content of insulin compound may be higher.

[0028] There is also described herein an insulin preparation comprising an
insulin compound, a nicotinic compound and glutamic acid.

[0031] The term "onset" refers to the time from injection until the PK
curve shifts to an increase.

[0032] The term "absorption rate" refers to the slope of the PK curve.

[0033] An "insulin compound" according to the invention is herein to be
understood as human insulin, an insulin analogue and/or any combination
thereof.

[0034] The term "human insulin" as used herein means the human hormone
whose structure and properties are well-known. Human insulin has two
polypeptide chains that are connected by disulphide bridges between
cysteine residues, namely the A-chain and the B-chain. The A-chain is a
21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two
chains being connected by three disulphide bridges: one between the
cysteines in position 6 and 11 of the A-chain, the second between the
cysteine in position 7 of the A-chain and the cysteine in position 7 of
the B-chain, and the third between the cysteine in position 20 of the
A-chain and the cysteine in position 19 of the B-chain.

[0035] The hormone is synthesized as a single-chain precursor proinsulin
(preproinsulin) consisting of a prepeptide of 24 amino acids followed by
proinsulin containing 86 amino acids in the configuration:
prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of
31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of
the connecting peptide from the A and B chains.

[0036] By "insulin analogue" as used herein is meant a polypeptide derived
from the primary structure of a naturally occurring insulin, for example
that of human insulin, by mutation. One or more mutations are made by
deleting and/or substituting at least one amino acid residue occurring in
the naturally occurring insulin and/or by adding at least one amino acid
residue. The added and/or substituted amino acid residues can either be
codable amino acid residues or other naturally occurring amino acid
residues.

[0037] In one embodiment an insulin analogue comprises less than 8
modifications (substitutions, deletions, additions and any combination
thereof) relative to the parent insulin, alternatively less than 7
modifications relative to the parent insulin, alternatively less than 6
modifications relative to the parent insulin, alternatively less than 5
modifications relative to the parent insulin, alternatively less than 4
modifications relative to the parent insulin, alternatively less than 3
modifications relative to the parent insulin, alternatively less than 2
modifications relative to the parent insulin.

[0038] Mutations in the insulin molecule are denoted stating the chain (A
or B), the position, and the three letter code for the amino acid
substituting the native amino acid. By "desB30" or "B(1-29)" is meant a
natural insulin B chain or analogue thereof lacking the B30 amino acid
residue, and by B28Asp human insulin is meant human insulin wherein the
amino acid residue in position 28 of the B chain has been substituted
with Asp.

[0039] Examples of insulin analogues are such wherein Pro in position 28
of the B chain is mutated with Asp, Lys, Leu, Val, or Ala and/or Lys at
position B29 is mutated with Pro, Glu or Asp. Furthermore, Asn at
position B3 may be mutated with Thr, Lys, Gln, Glu or Asp. The amino acid
residue in position A21 may be mutated with Gly. The amino acid in
position B1 may be mutated with Glu. The amino acid in position B16 may
be mutated with Glu or His. Further examples of insulin analogues are the
deletion analogues e.g. analogues where the B30 amino acid in human
insulin has been deleted (des(B30) human insulin), insulin analogues
wherein the B1 amino acid in human insulin has been deleted (des(B1)
human insulin), des(B28-B30) human insulin and des(B27) human insulin.
Insulin analogues wherein the A-chain and/or the B-chain have an
N-terminal extension and insulin analogues wherein the A-chain and/or the
B-chain have a C-terminal extension such as with two arginine residues
added to the C-terminal of the B-chain are also examples of insulin
analogues. Further examples are insulin analogues comprising combinations
of the mentioned mutations. Insulin analogues wherein the amino acid in
position A14 is Asn, Gln, Glu, Arg, Asp, Gly or His, the amino acid in
position B25 is His and which optionally further comprises one or more
additional mutations are further examples of insulin analogues. Insulin
analogues of human insulin wherein the amino acid residue in position A21
is Gly and wherein the insulin analogue is further extended in the
C-terminal with two arginine residues are also examples of insulin
analogues.

[0042] According to the present invention, the concentration of the
nicotinic compound and/or salts thereof is in the range from about 1 mM
to about 300 mM or from about 5 mM to about 200 mM.

[0043] The term "arginine" or "Arg" includes the amino acid arginine
and/or a salt thereof.

[0044] In one embodiment, the insulin preparation comprises 1 to 100 mM of
arginine.

[0045] In one embodiment, the insulin preparation comprises 1 to 20 mM of
arginine.

[0046] In one embodiment, the insulin preparation comprises 20 to 90 mM of
arginine.

[0047] In one embodiment, the insulin preparation comprises 30 to 85 mM of
arginine.

[0048] The term "glutamic acid" or "Glu" includes the aminoacid glutamic
acid and/or a salt thereof.

[0049] In one embodiment, the insulin preparation comprises 1 to 100 mM of
glutamic acid.

[0050] In one embodiment, the insulin preparation comprises 20 to 90 mM of
glutamic acid.

[0051] In one embodiment, the insulin preparation comprises 30 to 85 mM of
glutamic acid.

[0052] The term "pharmaceutical preparation" or "insulin preparation" as
used herein means a product comprising an insulin compound, i.e., a human
insulin, an analogue thereof and/or combinations thereof and a nicotinic
compound and an amino acid, optionally together with other excipients
such as preservatives, chelating agents, tonicity modifiers, bulking
agents, stabilizers, antioxidants, polymers and surfactants, metal ions,
oleaginous vehicles and proteins (e.g., human serum albumin, gelatine or
proteins), said insulin preparation being useful for treating, preventing
or reducing the severity of a disease or disorder by administration of
said insulin preparation to a person. Thus, an insulin preparation is
also known in the art as a pharmaceutical preparation or pharmaceutical
composition.

[0054] The insulin preparation of the present invention may further
comprise other ingredients common to insulin preparations, for example
zinc complexing agents such as citrate, and phosphate buffers.

[0055] Glycerol and/or mannitol and/or sodium chloride may be present in
an amount corresponding to a concentration of 0 to 250 mM, 0 to 200 mM or
0 to 100 mM.

[0056] Stabilizers, surfactants and preservatives may also be present in
the insulin preparations of this invention.

[0057] The insulin preparations of the present invention may further
comprise a pharmaceutically acceptable preservative. The preservative may
be present in an amount sufficient to obtain a preserving effect. The
amount of preservative in an insulin preparation may be determined from
e.g. literature in the field and/or the known amount(s) of preservative
in e.g. commercial products. Each one of these specific preservatives
constitutes an alternative embodiment of the invention. The use of a
preservative in pharmaceutical preparations is described, for example in
Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0058] The preservative present in the insulin preparation of this
invention may be as in the heretofore conventional insulin preparations,
for example phenol, m-cresol and methylparaben.

[0059] The insulin preparation of the present invention may further
comprise a chelating agent. The use of a chelating agent in
pharmaceutical preparations is well-known to the skilled person. For
convenience reference is made to Remington: The Science and Practice of
Pharmacy, 19th edition, 1995.

[0060] The insulin preparation of the present invention may further
comprise a stabilizer. The term "stabilizer" as used herein refers to
chemicals added to polypeptide containing pharmaceutical preparations in
order to stabilize the peptide, i.e. to increase the shelf life and/or
in-use time of such preparations. For convenience reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0061] The insulin preparation of the present invention may further
comprise a surfactant. The term "surfactant" as used herein refers to any
molecules or ions that are comprised of a water-soluble (hydrophilic)
part, the head, and a fat-soluble (lipophilic) segment. Surfactants
accumulate preferably at interfaces, which the hydrophilic part is
orientated towards the water (hydrophilic phase) and the lipophilic part
towards the oil- or hydrophobic phase (i.e. glass, air, oil etc.). The
concentration at which surfactants begin to form micelles is known as the
critical micelle concentration or CMC. Furthermore, surfactants lower the
surface tension of a liquid. Surfactants are also known as amphipathic
compounds. The term "detergent" is a synonym used for surfactants in
general. The use of a surfactant in pharmaceutical preparations is
well-known to the skilled person. For convenience reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0062] In a further embodiment the invention relates to an insulin
preparation comprising an aqueous solution of an insulin compound of the
present invention, and a buffer, wherein said insulin compound is present
in a concentration from 0.1 mM or above, and wherein said preparation has
a pH from about 3.0 to about 8.5 at room temperature (˜25°
C.).

[0063] The present invention also relates to methods for producing the
insulin preparations of the invention.

[0064] In one embodiment, the method for making insulin preparations of
the invention comprises:

[0065] a) preparing a solution by dissolving the insulin compound or a
mixture of insulin compounds in water or buffer;

[0066] b) preparing a solution by dissolving a divalent metal ion in water
or buffer;

[0067] c) preparing a solution by dissolving a preservative in water or
buffer;

[0068] d) preparing a solution by dissolving an isotonicity agent in water
or buffer;

[0069] e) preparing a solution by dissolving a surfactant and/or a
stabilizer in water or buffer;

[0070] f) mixing solution a) and one or more of solutions b), c), d), and
e);

[0071] Finally adjusting the pH of the mixture in f) to the desired pH
followed by a sterile filtration.

[0072] The insulin preparations of the present invention can be used in
the treatment of diabetes by parenteral administration. It is recommended
that the dosage of the insulin preparations of this invention which is to
be administered to the patient be selected by a physician.

[0073] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of a
syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. As a
further option, the insulin preparations containing the insulin compound
of the invention can also be adapted to transdermal administration, e.g.
by needle-free injection or from a patch, optionally an iontophoretic
patch, or transmucosal, e.g. buccal, administration.

[0074] Insulin preparations according to the present invention may be
administered to a patient in need of such treatment at several sites, for
example, at topical sites, for example, skin and mucosal sites, at sites
which bypass absorption, for example, administration in an artery, in a
vein, in the heart, and at sites which involve absorption, for example,
administration in the skin, under the skin, in a muscle or in the
abdomen.

[0075] In one embodiment of the invention the insulin preparation is an
aqueous preparation, i.e. preparation comprising water. Such preparation
is typically a solution or a suspension. In a further embodiment of the
invention the insulin preparation is an aqueous solution.

[0076] The term "aqueous preparation" is defined as a preparation
comprising at least 50% w/w water. Likewise, the term "aqueous solution"
is defined as a solution comprising at least 50% w/w water, and the term
"aqueous suspension" is defined as a suspension comprising at least 50%
w/w water.

[0077] Aqueous suspensions may contain the active compounds in admixture
with excipients suitable for the manufacture of aqueous suspensions.

[0078] In one embodiment, the insulin preparations of this invention are
well-suited for application in pen-like devices used for insulin therapy
by injection.

[0079] In one embodiment the insulin preparations of the present invention
can be used in pumps for insulin administration.

[0080] The term "physical stability" of the insulin preparation as used
herein refers to the tendency of the protein to form biologically
inactive and/or insoluble aggregates of the protein as a result of
exposure of the protein to thermo-mechanical stresses and/or interaction
with interfaces and surfaces that are destabilizing, such as hydrophobic
surfaces and interfaces. Physical stability of the aqueous protein
preparations is evaluated by means of visual inspection and/or turbidity
measurements after exposing the preparation filled in suitable containers
(e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual inspection of
the preparations is performed in a sharp focused light with a dark
background. The turbidity of the preparation is characterized by a visual
score ranking the degree of turbidity for instance on a scale from 0 to 3
(a preparation showing no turbidity corresponds to a visual score 0, and
a preparation showing visual turbidity in daylight corresponds to visual
score 3). A preparation is classified physically unstable with respect to
protein aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the preparation can be evaluated by
simple turbidity measurements well-known to the skilled person. Physical
stability of the aqueous protein preparations can also be evaluated by
using a spectroscopic agent or probe of the conformational status of the
protein. The probe is preferably a small molecule that preferentially
binds to a non-native conformer of the protein. One example of a small
molecular spectroscopic probe of protein structure is Thioflavin T.
Thioflavin T is a fluorescent dye that has been widely used for the
detection of amyloid fibrils. In the presence of fibrils, and perhaps
other protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about 482 nm
when bound to a fibril protein form. Unbound Thioflavin T is essentially
non-fluorescent at the wavelengths.

[0081] The term "chemical stability" of the protein preparation as used
herein refers to changes in the covalent protein structure leading to
formation of chemical degradation products with potential less biological
potency and/or potential increased immunogenic properties compared to the
native protein structure. Various chemical degradation products can be
formed depending on the type and nature of the native protein and the
environment to which the protein is exposed. Increasing amounts of
chemical degradation products is often seen during storage and use of the
protein preparation. Most proteins are prone to deamidation, a process in
which the side chain amide group in glutaminyl or asparaginyl residues is
hydrolysed to form a free carboxylic acid or asparaginyl residues to form
an IsoAsp derivative. Other degradations pathways involves formation of
high molecular weight products where two or more protein molecules are
covalently bound to each other through transamidation and/or disulfide
interactions leading to formation of covalently bound dimer, oligomer and
polymer degradation products (Stability of Protein Pharmaceuticals,
Ahern. T. J. & Manning M. C., Plenum Press, New York 1992). Oxidation (of
for instance methionine residues) can be mentioned as another variant of
chemical degradation. The chemical stability of the protein preparation
can be evaluated by measuring the amount of the chemical degradation
products at various time-points after exposure to different environmental
conditions (the formation of degradation products can often be
accelerated by for instance increasing temperature). The amount of each
individual degradation product is often determined by separation of the
degradation products depending on molecule size and/or charge using
various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC). Since
HMWP products are potentially immunogenic and not biologically active,
low levels of HMWP are advantageous.

[0082] The term "stabilized preparation" refers to a preparation with
increased physical stability, increased chemical stability or increased
physical and chemical stability. In general, a preparation must be stable
during use and storage (in compliance with recommended use and storage
conditions) until the expiration date is reached.

[0083] The term "diabetes" or "diabetes mellitus" includes type 1
diabetes, type 2 diabetes, gestational diabetes (during pregnancy) and
other states that cause hyperglycaemia. The term is used for a metabolic
disorder in which the pancreas produces insufficient amounts of insulin,
or in which the cells of the body fail to respond appropriately to
insulin thus preventing cells from absorbing glucose. As a result,
glucose builds up in the blood.

[0084] Type 1 diabetes, also called insulin-dependent diabetes mellitus
(IDDM) and juvenileonset diabetes, is caused by B-cell destruction,
usually leading to absolute insulin deficiency.

[0086] The term "pharmaceutically acceptable" as used herein means suited
for normal pharmaceutical applications, i.e., not giving rise to any
serious adverse events in patients.

[0087] The term "treatment of a disease" as used herein means the
management and care of a patient having developed the disease, condition
or disorder and includes treatment, prevention or alleviation of the
disease. The purpose of treatment is to combat the disease, condition or
disorder. Treatment includes the administration of the active compounds
to eliminate or control the disease, condition or disorder as well as to
alleviate the symptoms or complications associated with the disease,
condition or disorder, and prevention of the disease, condition or
disorder.

[0088] In another embodiment, an insulin analogue according to the
invention is used as a medicament for delaying or preventing disease
progression in type 2 diabetes.

[0089] In one embodiment of the present invention, the insulin preparation
according to the invention is for use as a medicament for the treatment
or prevention of hyperglycemia including stress induced hyperglycemia,
type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns,
operation wounds and other diseases or injuries where an anabolic effect
is needed in the treatment, myocardial infarction, stroke, coronary heart
disease and other cardiovascular disorders is provided.

[0090] In a further embodiment of the present invention, a method for the
treatment or prevention of hyperglycemia including stress induced
hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, and burns, operation wounds and other diseases or injuries
where an anabolic effect is needed in the treatment, myocardial
infarction, coronary heart disease and other cardiovascular disorders,
stroke, the method comprising administering to a patient in need of such
treatment an effective amount for such treatment of an insulin
preparation according to the invention, is provided.

[0091] The treatment with an insulin preparation according to the present
invention may also be combined with a second or more pharmacologically
active substances, e.g. selected from antidiabetic agents, antiobesity
agents, appetite regulating agents, antihypertensive agents, agents for
the treatment and/or prevention of complications resulting from or
associated with diabetes and agents for the treatment and/or prevention
of complications and disorders resulting from or associated with obesity.

[0092] The treatment with an insulin preparation according to the present
invention may also be combined with bariatric surgery--a surgery that
influences the glucose levels and/or lipid homeostasis such as gastric
banding or gastric bypass.

[0093] The production of polypeptides, e.g., insulins, is well known in
the art. An insulin analogue according to the invention may for instance
be produced by classical peptide synthesis, e.g. solid phase peptide
synthesis using t-Boc or Fmoc chemistry or other well established
techniques, see e.g. Greene and Wuts, "Protective Groups in Organic
Synthesis", John Wiley & Sons, 1999. The insulin analogue may also be
produced by a method which comprises culturing a host cell containing a
DNA sequence encoding the analogue and capable of expressing the insulin
analogue in a suitable nutrient medium under conditions permitting the
expression of the insulin analogue. For insulin analogues comprising
non-natural amino acid residues, the recombinant cell should be modified
such that the non-natural amino acids are incorporated into the analogue,
for instance by use of tRNA mutants. Hence, briefly, the insulin
analogues according to the invention are prepared analogously to the
preparation of known insulin analogues.

[0094] Several methods may be used for the production of human insulin and
human insulin analogues. For example three major methods which are used
in the production of insulin in microorganisms are disclosed in
WO2008034881. Two of these involve Escherichia coli, with either the
expression of a large fusion protein in the cytoplasm (Frank et al.
(1981) in Peptides: Proceedings of the 7th American Peptide
Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford,
Ill. pp 729-739), or use of a signal peptide to enable secretion into the
periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method
utilizes Saccharomyces cerevisiae to secrete an insulin precursor into
the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art
discloses a number of insulin precursors which are expressed in either E.
coli or Saccharomyces cerevisiae, vide U.S. Pat. No. 5,962,267, WO
95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

[0095] The insulin analogues are produced by expressing a DNA sequence
encoding the insulin analogue in question in a suitable host cell by well
known technique as disclosed in e.g. U.S. Pat. No. 6,500,645. The insulin
analogue is either expressed directly or as a precursor molecule which
has an N-terminal extension on the B-chain or a C-terminal extension on
the B-chain. The N-terminal extension may have the function of increasing
the yield of the directly expressed product and may be of up to 15 amino
acid residues long. The N-terminal extension is to be cleaved of in vitro
after isolation from the culture broth and will therefore have a cleavage
site next to B1. N-terminal extensions of the type suitable in the
present invention are disclosed in U.S. Pat. No. 5,395,922, and EP
765,395. The C-terminal extension may have the function of protecting the
mature insulin or insulin analogue molecule against intracellular
proteolytic processing by host cell exoproteases. The C-terminal
extension is to be cleaved of either extra-cellularly in the culture
broth by secreted, active carboxypeptidase or in vitro after isolation
from the culture broth. A method for producing mature insulin and insulin
analogs with C-terminal extensions on the B-chain that are removed by
carboxypetidase are disclosed in WO 08037735. The target insulin product
of the process may either be a two-chain human insulin or a two-chain
human insulin analogue which may or may not have a short C-terminal
extension of the B-chain. If the target insulin product will have no
C-terminal extension of the B-chain, then said C-terminal extension
should be capable of subsequently being cleaved off from the B-chain
before further purification steps.

[0096] The present invention also contemplates the following non-limiting
list of embodiments, which are further described elsewhere herein: [0097]
1. An insulin preparation comprising:

[0098] an insulin compound,

[0099] a nicotinic compound, and

[0100] arginine. [0101] 2. The insulin preparation according to embodiment
1, wherein the insulin compound is human insulin or an insulin analog.
[0102] 3. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is B28Asp human insulin. [0103]
4. The insulin preparation according to any of the preceding embodiments,
wherein the insulin compound is B28LysB29Pro human insulin. [0104] 5. The
insulin preparation according to any of the preceding embodiments,
wherein the insulin compound is B3LysB29Glu human insulin. [0105] 6. The
insulin preparation according to any of the preceding embodiments,
wherein the insulin compound is present in a range selected from the
following: 0.1-10.0 mM; 0.1-3.0 mM; 0.1-2.5 mM; 0.1-2.0 mM; 0.1-1.5 mM;
0.2-2.5 mM; 0.2-2.0 mM; 0.2-1.5 mM; 0.3-3.0 mM; 0.3-2.5 mM; 0.3-2.0 mM;
0.3-1.5 mM; 0.5-1.3 mM and 0.6-1.2 mM. [0106] 7. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in the amount from about 0.1 mM to about 10.0 mM.
[0107] 8. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount from
about 0.1 mM to about 3.0 mM. [0108] 9. The insulin preparation according
to any of the preceding embodiments, wherein the insulin compound is
present in the amount from about 0.1 mM to about 2.5 mM. [0109] 10. The
insulin preparation according to any of the preceding embodiments,
wherein the insulin compound is present in the amount from about 0.1 mM
to about 2.0 mM. [0110] 11. The insulin preparation according to any of
the preceding embodiments, wherein the insulin compound is present in the
amount from about 0.1 mM to about 1.5 mM. [0111] 12. The insulin
preparation according to any of the preceding embodiments, wherein the
insulin compound is present in the amount from about 0.2 mM to about 2.5
mM. [0112] 13. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount from
about 0.2 mM to about 2.0 mM. [0113] 14. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in the amount from about 0.2 mM to about 1.5 mM.
[0114] 15. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount from
about 0.3 mM to about 3.0 mM. [0115] 16. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in the amount from about 0.3 mM to about 2.5 mM.
[0116] 17. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount from
about 0.3 mM to about 2.0 mM. [0117] 18. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in the amount from about 0.3 mM to about 1.5 mM.
[0118] 19. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount from
about 0.5 mM to about 1.3 mM. [0119] 20. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in the amount from about 0.3 mM to about 1.2 mM.
[0120] 21. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount from
about 0.6 mM to about 1.2 mM. [0121] 22. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in the amount of about 0.6 or about 1.2 mM. [0122]
23. The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount of
about 0.3 mM. [0123] 24. The insulin preparation according to any of the
preceding embodiments, wherein the insulin compound is present in the
amount of about 0.6 mM. [0124] 25. The insulin preparation according to
any of the preceding embodiments, wherein the insulin compound is present
in the amount of about 1.2 mM. [0125] 26. The insulin preparation
according to any of the preceding embodiments, wherein the nicotinic
compound is selected from the group consisting of nicotinamide, nicotinic
acid, niacin, niacin amide and vitamin B3 and/or salts thereof and/or any
combination thereof. [0126] 27. The insulin preparation according to any
of the preceding embodiments, wherein the nicotinic compound is selected
from nicotinamide and nicotinic acid and/or salts thereof and/or any
combination thereof. [0127] 28. The insulin preparation according to any
of the preceding embodiments, wherein the nicotinic compound is present
in a range selected from the following: 1-300 mM; 5-200 mM; 40-120 mM,
70-140 mM or 80-130 mM. [0128] 29. The insulin preparation according to
any of the preceding embodiments, comprising from about 1 mM to about 300
mM of the nicotinic compound. [0129] 30. The insulin preparation
according to any of the preceding embodiments, comprising from about 8 mM
to about 260 mM of the nicotinic compound. [0130] 31. The insulin
preparation according to any of the preceding embodiments, comprising
from about 5 mM to about 200 mM of the nicotinic compound. [0131] 32. The
insulin preparation according to any of the preceding embodiments,
comprising from about 1 mM to about 150 mM of the nicotinic compound.
[0132] 33. The insulin preparation according to any of the preceding
embodiments, comprising from about 5 mM to about 20 mM of the nicotinic
compound. [0133] 34. The insulin preparation according to any of the
preceding embodiments, comprising from about 20 mM to about 120 mM of the
nicotinic compound. [0134] 35. The insulin preparation according to any
of the preceding embodiments, comprising from about 40 mM to about 120 mM
of the nicotinic compound. [0135] 36. The insulin preparation according
to any of the preceding embodiments, comprising from about 20 mM to about
40 mM of the nicotinic compound. [0136] 37. The insulin preparation
according to any of the preceding embodiments, comprising from about 60
mM to about 80 mM of the nicotinic compound. [0137] 38. The insulin
preparation according to any of the preceding embodiments, comprising
from about 70 mM to about 140 mM of the nicotinic compound. [0138] 39.
The insulin preparation according to any of the preceding embodiments,
comprising from about 80 mM to about 130 mM of the nicotinic compound.
[0139] 40. The insulin preparation according to any of the preceding
embodiments, comprising about 8 mM, 30 mM, 100 mM or 130 mM of the
nicotinic compound. [0140] 41. The insulin preparation according to any
of the preceding embodiments, comprising about 8 mM of the nicotinic
compound. [0141] 42. The insulin preparation according to any of the
preceding embodiments, comprising about 30 mM, 100 mM or 130 mM of the
nicotinic compound. [0142] 43. The insulin preparation according to any
of the preceding embodiments, comprising about 30 mM of the nicotinic
compound. [0143] 44. The insulin preparation according to any of the
preceding embodiments, comprising about 100 mM of the nicotinic compound.
[0144] 45. The insulin preparation according to any of the preceding
embodiments, comprising about 130 mM of the nicotinic compound. [0145]
46. The insulin preparation according to any of the preceding
embodiments, comprising about 150 mM of the nicotinic compound. [0146]
47. The insulin preparation according to any of the preceding
embodiments, comprising the following ranges of arginine compound: 1-100
mM, 5-120 mM, 8-85 mM, 20-90 mM, 30-90 mM, 30-85 mM, 30-60 mM or 10-40
mM. [0147] 48. The insulin preparation according to any of the preceding
embodiments, comprising the following ranges of arginine compound: 1-120
mM, 8-85 mM or 1-40 mM. [0148] 49. The insulin preparation according to
any of the preceding embodiments, comprising from about 1 mM to about 120
mM of arginine. [0149] 50. The insulin preparation according to any of
the preceding embodiments, comprising from about 1 mM to about 100 mM of
arginine. [0150] 51. The insulin preparation according to any of the
preceding embodiments, comprising from about 5 mM to about 120 mM of
arginine. [0151] 52. The insulin preparation according to any of the
preceding embodiments, comprising from about 20 mM to about 90 mM of
arginine. [0152] 53. The insulin preparation according to any of the
preceding embodiments, comprising from about 30 mM to about 85 mM of
arginine. [0153] 54. The insulin preparation according to any of the
preceding embodiments, comprising from about 8 mM to about 85 mM of
arginine. [0154] 55. The insulin preparation according to any of the
preceding embodiments, comprising from about 30 mM to about 60 mM of
arginine. [0155] 56. The insulin preparation according to any of the
preceding embodiments, comprising from about 10 mM to about 40 mM of
arginine. [0156] 57. The insulin preparation according to any of the
preceding embodiments, comprising from about 1 mM to about 40 mM of
arginine. [0157] 58. The insulin preparation according to any of the
preceding embodiments, wherein arginine is present in a range selected
from the following: 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM,
10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mM, 45 mM, 50 mM, 55 mM or
60 mM. [0158] 59. The insulin preparation according to any of the
preceding embodiments, comprising about 1 mM of arginine. [0159] 60. The
insulin preparation according to any of the preceding embodiments,
comprising about 2 mM of arginine. [0160] 61. The insulin preparation
according to any of the preceding embodiments, comprising about 3 mM of
arginine. [0161] 62. The insulin preparation according to any of the
preceding embodiments, comprising about 4 mM of arginine. [0162] 63. The
insulin preparation according to any of the preceding embodiments,
comprising about 5 mM of arginine. [0163] 64. The insulin preparation
according to any of the preceding embodiments, comprising about 6 mM of
arginine. [0164] 65. The insulin preparation according to any of the
preceding embodiments, comprising about 7 mM of arginine. [0165] 66. The
insulin preparation according to any of the preceding embodiments,
comprising about 8 mM of arginine. [0166] 67. The insulin preparation
according to any of the preceding embodiments, comprising about 9 mM of
arginine. [0167] 68. The insulin preparation according to any of the
preceding embodiments, comprising about 10 mM of arginine. [0168] 69. The
insulin preparation according to any of the preceding embodiments,
comprising about 15 mM of arginine. [0169] 70. The insulin preparation
according to any of the preceding embodiments, comprising about 20 mM of
arginine. [0170] 71. The insulin preparation according to any of the
preceding embodiments, comprising about 25 mM of arginine. [0171] 72. The
insulin preparation according to any of the preceding embodiments,
comprising about 30 mM of arginine. [0172] 73. The insulin preparation
according to any of the preceding embodiments, comprising about 35 mM of
arginine. [0173] 74. The insulin preparation according to any of the
preceding embodiments, comprising about 40 mM of arginine. [0174] 75. The
insulin preparation according to any of the preceding embodiments,
comprising about 45 mM of arginine. [0175] 76. The insulin preparation
according to any of the preceding embodiments, comprising about 50 mM of
arginine. [0176] 77. The insulin preparation according to any of the
preceding embodiments, comprising about 55 mM of arginine. [0177] 78. The
insulin preparation according to any of the preceding embodiments,
comprising about 60 mM of arginine. [0178] 79. The insulin preparation
according to any of the preceding embodiments, further comprising
glutamic acid. [0179] 80. The insulin preparation according to embodiment
79, wherein glutamic acid is present in a range selected from the
following: 1-100 mM, 20-90 mM, 30-90 mM, 30-85 mM or 30-50 mM. [0180] 81.
The insulin preparation according to embodiment 79, comprising from about
1 mM to about 100 mM of glutamic acid. [0181] 82. The insulin preparation
according to embodiment 79, comprising from about 20 mM to about 90 mM of
glutamic acid. [0182] 83. The insulin preparation according to embodiment
79, comprising from about 30 mM to about 85 mM of glutamic acid. [0183]
84. The insulin preparation according to embodiment 79, comprising from
about 30 mM to about 50 mM of glutamic acid. [0184] 85. The insulin
preparation according to embodiment 79, comprising about 30 mM or 50 mM
of glutamic acid. [0185] 86. The insulin preparation according to
embodiment 79, comprising about 30 mM of glutamic acid. [0186] 87. The
insulin preparation according to embodiment 79, comprising about 50 mM of
glutamic acid. [0187] 88. The insulin preparation according to any of the
preceding embodiments, which further comprises a metal ion, preservative
agent(s), isotonicity agent(s) and stabilizer(s), detergent(s), and
buffer(s). [0188] 89. The insulin preparation according to embodiment 88,
wherein said buffer is Tris. [0189] 90. The insulin preparation according
to embodiment 89, comprising from about 2 mM to about 50 mM of Tris.
[0190] 91. The insulin preparation according to embodiment 89, comprising
from about 10 mM to about 40 mM of Tris. [0191] 92. The insulin
preparation according to embodiment 89, comprising from about 20 mM to
about 30 mM of Tris. [0192] 93. The insulin preparation according to
embodiment 89, comprising about 10 mM, 20 mM, 30 mM or 40 mM of Tris.
[0193] 94. The insulin preparation according to embodiment 89, comprising
about 10 mM of Tris. [0194] 95. The insulin preparation according to
embodiment 89, comprising about 20 mM of Tris. [0195] 96. The insulin
preparation according to embodiment 89, comprising about 30 mM of Tris.
[0196] 97. The insulin preparation according to embodiment 89, comprising
about 40 mM of Tris. [0197] 98. The insulin preparation according to
embodiment 89, wherein the metal ion is zinc. [0198] 99. The insulin
preparation according to embodiment 98, wherein less than about 6 zinc
ions are present per hexamer of insulin compound. [0199] 100. The insulin
preparation according to embodiment 98, wherein less than about 4 zinc
ions are present per hexamer of insulin compound. [0200] 101. The insulin
preparation according to embodiment 98, wherein less than about 3 zinc
ions are present per hexamer of insulin compound. [0201] 102. The insulin
preparation according to embodiment 98, wherein the zinc:insulin molar
ratio is from about 2:6 to about 5:6. [0202] 103. The insulin preparation
according to embodiment 98, wherein the zinc:insulin molar ratio is from
about 2.5:6 to about 4.5:6. [0203] 104. The insulin preparation according
to embodiment 98, wherein the zinc:insulin molar ratio is from about 3:6
to about 4:6. [0204] 105. The insulin preparation according to embodiment
98, wherein the zinc:insulin molar ratio is about 2:6.

[0205] 106. The insulin preparation according to embodiment 98, wherein
the zinc:insulin molar ratio is about 2.5:6. [0206] 107. The insulin
preparation according to embodiment 98, wherein the zinc:insulin molar
ratio is about 3:6. [0207] 108. The insulin preparation according to
embodiment 98, wherein the zinc:insulin molar ratio is about 3.5:6.
[0208] 109. The insulin preparation according to embodiment 98, wherein
the zinc:insulin molar ratio is about 4:6. [0209] 110. The insulin
preparation according to embodiment 98, wherein the zinc:insulin molar
ratio is about 4.5:6. [0210] 111. The insulin preparation according to
embodiment 98, wherein the zinc:insulin molar ratio is about 5:6. [0211]
112. The insulin preparation according to embodiment 88, wherein the
stabilizer is a non-ionic detergent. [0212] 113. The insulin preparation
according to embodiment 112, wherein the detergent is polysorbate 20
(Tween 20) or polysorbate 80 (Tween 80). [0213] 114. The insulin
preparation according to embodiment 112, wherein the detergent is
polysorbate 20 (Tween 20). [0214] 115. The insulin preparation according
to embodiment 112, wherein the detergent is polysorbate 80 (Tween 80).
[0215] 116. The insulin preparation according to any of embodiments
112-115, comprising from about 5 to 100 ppm, from about 10 to about 50
ppm or from about 10 to about 20 ppm of polysorbate. [0216] 117. The
insulin preparation according to embodiment 88, further comprising a
phenolic compound. [0217] 118. The insulin preparation according to
embodiment 117, wherein said phenolic compound is present in the amount
from about 0 to about 6 mg/ml or from about 0 to about 4 mg/ml. [0218]
119. The insulin preparation according to embodiment 88, further
comprising m-cresol. [0219] 120. The insulin preparation according to
embodiment 119, wherein m-cresol is present in the amount from about 0.5
to about 4.0 mg/ml or from about 0.6 to about 4.0 mg/ml. [0220] 121. An
insulin preparation according to any of the previous embodiments, wherein
the pH is neutral to weakly basic. [0221] 122. An insulin preparation
according to any of the previous embodiments, wherein the pH is from
about 7.0 to about 8.0. [0222] 123. An insulin preparation according to
any of the previous embodiments, wherein the pH is about 7.0. [0223] 124.
An insulin preparation according to any of the previous embodiments,
wherein the pH is about 7.1. [0224] 125. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 7.2. [0225]
126. An insulin preparation according to any of the previous embodiments,
wherein the pH is about 7.3. [0226] 127. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 7.4. [0227]
128. An insulin preparation according to any of the previous embodiments,
wherein the pH is about 7.5. [0228] 129. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 7.6. [0229]
130. An insulin preparation according to any of the previous embodiments,
wherein the pH is about 7.7. [0230] 131. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 7.8. [0231]
132. An insulin preparation according to any of the previous embodiments,
wherein the pH is about 7.9. [0232] 133. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 8.0. [0233]
134. A method of reducing the blood glucose level in mammals by
administering to a patient in need of such treatment a therapeutically
active dose of an insulin preparation according to any of the preceding
embodiments. [0234] 135. A method for the treatment of diabetes mellitus
in a subject comprising administering to a subject an insulin preparation
according to any of the preceding embodiments. [0235] 136. A method
according to any of the preceding embodiments, for parenteral
administration. [0236] 137. An insulin preparation according to any of
the preceding embodiments, for use in the treatment or prevention of
hyperglycemia including stress induced hyperglycemia, type 2 diabetes,
impaired glucose tolerance, type 1 diabetes, and burns, operation wounds
and other diseases or injuries where an anabolic effect is needed in the
treatment, myocardial infarction, stroke, coronary heart disease and
other cardiovascular disorders and treatment of critically ill diabetic
and non-diabetic patients.

[0237] The invention is further illustrated by the following examples
which are not to be construed as limiting.

[0238] All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference in their
entirety and to the same extent as if each reference were individually
and specifically indicated to be incorporated by reference and were set
forth in its entirety herein (to the maximum extent permitted by law).

[0239] All headings and sub-headings are used herein for convenience only
and should not be construed as limiting the invention in any way.

[0240] The use of any and all examples, or exemplary language (e.g., "such
as") provided herein, is intended merely to better illuminate the
invention and does not pose a limitation on the scope of the invention
unless otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential to the
practice of the invention.

[0241] The citation and incorporation of patent documents herein is done
for convenience only and does not reflect any view of the validity,
patentability, and/or enforceability of such patent documents.

[0242] This invention includes all modifications and equivalents of the
subject matter recited in the claims appended hereto as permitted by
applicable law.

EXAMPLES

Example 1

Preparation of Pharmaceutical Preparations

[0243] The pharmaceutical preparations of the present invention may be
formulated as an aqueous solution. The aqueous medium is made isotonic,
for example, with sodium chloride or glycerol. Furthermore, the aqueous
medium may contain zinc ions, for example added as zinc acetate or zinc
chloride, buffers and preservatives. Arginine may be added as Arg, HCl.
The pH value of the preparation is adjusted to the desired value and may
be between about 3 to about 8.5, between about 3 and about 5 or about 6.5
and about 7.5 depending on the isoelectric point, pI, of the insulin in
question.

[0244] Quantitative determination of high molecular weight protein (HMWP)
and monomer insulin aspart was performed on Waters insulin (300×7.8
mm, part nr wat 201549) with an eluent containing 2.5M acetic acid, 4 mM
L-arginine and 20% (V/V) acetonitrile at a flow rate of 1 ml/min. and
40° C. Detection was performed with a tuneable absorbance detector
(Waters 486) at 276 nm. Injection volume was 40 μl and a 600 μM
human insulin standard. HMWP and concentration of the preparations were
measured at each sampling point.

Reverse Phase Chromatography (HPLC)

[0245] Determination of the insulin aspart related impurities were
performed on a HPLC system using a BEH RP C8 2.1×100 mm column,
particle size of 1.7 μm. Waters part no 186002878, with a flow rate of
0.5 ml/min., at 40° C. detection at 220 nm. Elution was performed
with a mobile phase consisting of the following:

[0248] The amount of B28 iso-aspartate, desamido and other related
impurities were determined as absorbance area measured in percent of
total absorbance area determined after elution of the preservatives. The
RP-HPLC method is equivalent to the analytical method used for quality
control of Novo Nordisk marketed insulin aspart pharmaceuticals.

[0249] Addition of arginine reduces the amount of degradation products
formed, especially HMWP and des-amido forms, increasing the concentration
of arginine in the range 10 to 50 mM leads to further reduction of
degradation. The physical stability measured as lag time in the ThT assay
is reduced upon addition of arginine and is increasingly reduced when the
arginine concentration is increased. The overall performance of 50 mM
arginine is superior to 50 mM glycine, 50 mM glutamic acid, or 50 mM
histidine regarding reduction of the formation of degradation products,
as is shown in Table 3 below.

[0250] The PK/PD studies were performed on domestic female pigs, LYD
cross-breed, weighing between 55 and 110 kg. The pigs were catheterised
into the jugular vein through an ear vein at least 2 days before start of
the study. The last meal before the start of the study was served to the
animals approx. 18 hours prior to the injection of the test preparation,
and the animals had free access to water at all time during the fasting
period and the test period.

[0251] At time 0 hours the test preparation was given subcutaneous on the
lateral side of the neck. A blood sample was drawn prior dosing and at
regular time intervals after dosing samples were drawn from the catheter
and sampled into 1.5 ml glass tubes pre-coated with heparin. The blood
samples were kept in ice water until separation of plasma by
centrifugetion for 10 min. 3000 rpm at 4° C., which was done
within the first 30 minutes. Plasma samples were stored at 4° C.
for short time (2-3 hours) or at -18° C. for long term storage and
were analysed for glucose on YSI or Konelab 30i and for insulin Aspart
concentration by LOCI.

[0252] The insulin Aspart LOCI is a monoclonal antibody-based sandwich
immunoassay and applies the proximity of two beads, the europium-coated
acceptor beads and the streptavidin coated donor-beads. The acceptor
beads were coated with a specific antibody against human insulin and
recognize insulin Aspart in plasma samples. A second biotinylated
antibody bind specific to insulin Aspart and together with the
streptavidin coated beads, they make up the sandwich. Illumination of the
beads-aggregate-immunocomplex releases singlet oxygen from the donor
beads which channels into the acceptor beads and triggers
chemiluminescence. The chemiluminescence was measured and the amount of
light generated is proportional to the concentration of insulin Aspart.

[0253] Compared to the marketed product NovoRapid®, the initial rate
of plasma glucose lowering is faster for the preparations of the present
invention (FIGS. 3 and 4). Likewise, when compared to NovoRapid®, the
initial absorption rate of the insulin component of the preparations of
the present invention, is markedly faster (FIG. 5).

Example 4

General Introduction to ThT Fibrillation Assays for the Assessment of
Physical Stability of Protein Formulations

[0254] Low physical stability of a peptide may lead to amyloid fibril
formation, which is observed as well-ordered, thread-like macromolecular
structures in the sample eventually resulting in gel formation. This has
traditionally been measured by visual inspection of the sample. However,
that kind of measurement is very subjective and depending on the
observer. Therefore, the application of a small molecule indicator probe
is much more advantageous. Thioflavin T (ThT) is such a probe and has a
distinct fluorescence signature when binding to fibrils [Naiki et al.
(1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309,
274-284].

[0255] The time course for fibril formation can be described by a
sigmoidal curve (FIG. 6) with the following expression [Nielsen et al.
(2001) Biochemistry 40, 6036-6046]:

[0256] Here, F is the ThT fluorescence at the time t. The constant t0
is the time needed to reach 50% of maximum fluorescence. The two
important parameters describing fibril form ation are the lag-time
calculated by t0-2τ and the apparent rate constant
kapp=1/τ.

[0257] Formation of a partially folded intermediate of the peptide is
suggested as a general initiating mechanism for fibrillation. Few of
those intermediates nucleate to form a template onto which further
intermediates may assembly and the fibrillation proceeds. The lag-time
corresponds to the interval in which the critical mass of nucleus is
built up and the apparent rate constant is the rate with which the fibril
itself is formed.

Sample Preparation

[0258] Samples were prepared freshly before each assay. Each sample
composition is described in each example. The pH of the sample was
adjusted to the desired value using appropriate amounts of concentrated
NaOH and HClO4 or HCl. Thioflavin T was added to the samples from a
stock solution in H2O to a final concentration of 1 μM.

[0259] Sample aliquots of 200 μl were placed in a 96 well microtiter
plate (Packard OptiPlate®-96, white polystyrene). Usually, four or
eight replica of each sample (corresponding to one test condition) were
placed in one column of wells. The plate was sealed with Scotch Pad
(Qiagen).

Incubation and Fluorescence Measurement

[0260] Incubation at given temperature, shaking and measurement of the ThT
fluorescence emission were done in a Fluoroskan Ascent FL fluorescence
platereader or Varioskan platereader (Thermo Labsystems). The temperature
was adjusted to 37° C. The orbital shaking was adjusted to 960 rpm
with an amplitude of 1 mm in all the presented data. Fluorescence
measurement was done using excitation through a 444 nm filter and
measurement of emission through a 485 nm filter.

[0261] Each run was initiated by incubating the plate at the assay
temperature for 10 min. The plate was measured every 20 minutes for a
desired period of time. Between each measurement, the plate was shaken
and heated as described.

Data Handling

[0262] The measurement points were saved in Microsoft Excel format for
further processing and curve drawing and fitting was performed using
Graph Pad Prism. The background emission from ThT in the absence of
fibrils was negligible. The data points are typically a mean of four or
eight samples and shown with standard deviation error bars. Only data
obtained in the same experiment (i.e. samples on the same plate) are
presented in the same graph ensuring a relative measure of fibrillation
between experiments.

[0263] The data set may be fitted to Eq. (1). However, since full
sigmodial curves are not always achieved during the measurement time, lag
times were here visually determined from the ThT fluorescence curve as
the time point at which the ThT fluorescence is different than the
background level.

Measurement of Initial and Final Concentrations

[0264] The peptide concentration in each of the tested formulations were
measured both before application in the ThT fibrillation assay
("Initial") and after completion of the ThT fibrillation ("After ThT
assay"). Concentrations were determined by reverse HPLC methods using a
pramlintide standard as a reference. Before measurement after completion
150 μl was collected from each of the replica and transferred to an
Eppendorf tube. These were centrifuged at 30000 G for 40 mins. The
supernatants were filtered through a 0.22 μm filter before application
on the HPLC system.